Abstract Defatting and dry fractionation of soybean flour by the combination of impact milling and tribo-electrostatic separation was investigated to prepare proteinâenriched soybean flour. Defatting is crucial to facilitate dry milling required for dry fractionation. Both organic solvent extraction and oil pressing were suitable as defatting methods although oil pressing compacted the tissue structure visually. Moderate impact milling (classifier wheel speed of 3000 rpm) effectively liberated protein bodies while eliminating agglomeration of small particles. Electrostatic separation was conducted with varying charging tube configurations. We found higher yields in the separation with a spiral charging tube design than with a slit design. The soy flour was enriched in protein from 37 g/100 g to 45 g/100 g dry basis by defatting. During electrostatic separation a soy protein enrichment of 15% was achieved and a yield of 62% of the protein was recovered from the defatted soy flour. Industrial applications The production of soy protein isolates and soy protein concentrates with wet fractionation consumes a lot of water and energy. Meanwhile, the ingredients lose their native state and part of their functionality due to the harsh processing conditions. This study proposed a dry fractionation method combining fine milling and electrostatic separation for the preparation of soybean protein-enriched fractions retaining the native state of the protein. Although the proposed dry fractionation for protein-enriched fractions from oilseeds is promising, still challenges are ahead for upscaling the process, being efficient and safe in its operation.